Project Summary/Abstract Stroke is a prevalent and devastating disease with limited therapeutic options. Inflammation and immune cells are major components in the pathophysiology of ischemic stroke and contribute to acute and delayed tissue injury. However, our incomplete understanding of the factors regulating the immune responses triggered by cerebral ischemia remains a significant obstacle to the development of effective therapeutic interventions based on modulating post-ischemic inflammation. Besides activation of brain resident immune cells, ischemic stroke is characterized by the recruitment of peripheral innate and adaptive immune cells that participate in the inflammatory response and contribute to the damage. Monocyte-derived macrophages (MM) are a major component of the cellular innate immune response to ischemic stroke. MM infiltrate the ischemic lesion and perilesional territories early after ischemia and reside in the injured territory for prolonged periods of time. These observations raise the possibility that MM contribute to the entire inflammatory process from initiation to resolution with the potential of modulating the outcome of cerebral ischemic injury. The long-term goal of this research program is to elucidate the role of MM in stroke pathophysiology and develop the experimental framework for new preventative and therapeutic approaches for ischemic stroke. In the present application, we will test the hypothesis that MM are present in the ischemic territory as functionally divergent and molecularly definable populations and that tissue hypoxia is an important determinator for MM differentiation and revelation of their neuroprotective capacity. This hypothesis, supported by relevant preliminary results, will be tested by determining: (a) the transition of MM from monocytes to long-term resident immune cells in the post-ischemic brain (Aim 1), (b) the effects of the hypoxic environment on tissue distribution and polarization of MM (Aim 2), and (c) the role of hypoxia-inducible factor 1? (HIF1?) and glycolytic metabolism in the functional polarization of brain-infiltrating MM and their effects on stroke outcome (Aim 3). These goals will be achieved using a mouse model of transient focal cerebral ischemia with assessment of histological and neurological outcome together with a novel model for classifying brain MM. This proposal may open the way to new avenues for stroke prevention and therapy based on modulation of MM and their precursor, the peripheral blood monocyte.